A role for tissue transglutaminase in alpha-gliadin peptide cytotoxicity

In coeliac disease, gliadin peptides p56-88, p57-68 and p31-49 have been demonstrated to be involved in the pathogenic damage of the small intestine via their immunogenicity or toxicity to epithelial cells. To try to understand the mechanism of their toxicity, we investigated the effect of synthetic peptides (p31-49, p56-88, p57-68, p69-82) and of their deamidated analogues on Caco2 and FHs 74 Int cell toxicity and tissue tranglutaminase activity. Apoptosis, necrosis and cell viability were assessed by flow cytometry, and peptide deamidation was determined indirectly by measuring its capacity to inhibit tTG activity. The results showed that p56-88 and p57-68 reduced cell growth and concomitantly inhibited tTG activity in both cell types. This effect was abolished when Caco2 cells were treated with antibodies to tTG. Deamidated peptide p57-68 (E(65)) lost practically all of its inhibitory effect on cell growth and on tTG activity. Cellular toxicity was also observed with p31-49, which was not a substrate for tTG. p69-82 was not cytotoxic but became so when glutamine 72 was substituted by glutamic acid. These findings provide evidence for the existence of three types of toxicity among gliadin peptides: (i) peptides that are intrinsically toxic and are not substrates of tTG; (ii) peptides that are non-toxic but become so when they act as substrates of tTG; and (iii) peptides that are non-toxic and are not substrates of tTG but become so when deamidated. A mechanism other than that involving tTG could be responsible for the deamidation of glutamine residues of gliadin in the intestinal tract.     

The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock

A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins, which is highly dependent on casein kinase Idelta/epsilon (CKIdelta/epsilon; termed DOUBLETIME [DBT] in Drosophila) and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, beta-TrCP (SLIMB in Drosophila). Here we use the Drosophila melanogaster model system, and show that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical SLIMB-binding site on PER. DBT-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located DBT docking site on PER and partially counterbalanced by protein phosphatase activity. We propose that the gradual DBT-mediated phosphorylation of a nonconsensus SLIMB-binding site establishes a temporal threshold for when in a daily cycle the majority of PER proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on PER stability. We also use mass spectrometry to map phosphorylation sites on PER, leading to the identification of a number of "phospho-clusters" that explain several of the classic per mutants.     

Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a “genetically deamidated” form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity.     

Evidence for age-related deamidation reactions in erythrocyte p55 and glycophorins C and D: implications for signal transduction involving tumour-suppressor proteins in higher eukaryotes

The methylation of erythrocyte cytoskeletal proteins by methyltransferases is a strategy for the repair of deamidated asparagine residues. Evidence from extant data shows that the junction point proteins of the erythrocyte cytoskeleton, band 3, ankyrin, glycophorins C/D, band 4.1, calmodulin, and p55 are all methylated in situ. It is suggested that sequence-dependent deamidation of asparagine residues in p55, 4.1 and glycophorins C/D could affect the interactions of these junction point proteins in an age-dependent manner. Comparison of the asparagine content of 4.1 binding regions of p55 analogues dig and hdlg acts as a caveat to the extrapolation of findings made in erythrocytes to other eukaryotic cells, and has important implications for transmembrane signalling pathways and age-dependent changes in the membrane-binding characteristics of tumour-suppressor proteins in higher cell systems.     

Pathomechanisms in celiac disease

Celiac disease is a complex autoimmune disease which is characterized by a strong genetic association (HLA-DQ2 or -DQ8), gluten as nutritional etiological factor, and the enzyme tissue transglutaminase as endomysial autoantigen. Patients develop highly predictive IgA autoantibodies to tTG. Certain gluten peptides are presented by the disease-associated HLA-DQ2/DQ8 molecules leading to stimulation of gluten-specific T cells. This immune response which is driven in the lamina propria causes the mucosal transformation characteristic for celiac disease. Increased intestinal expression of tTG in patients with CD appears to play an important role in the pathogenesis of CD. Thus, modification of gluten peptides by tTG, especially deamidation of certain glutamine residues, can enhance their binding to HLA-DQ2 or -DQ8 and potentiate T cell stimulation. Furthermore, tTG-catalyzed cross-linking and consequent haptenization of gluten with extracellular matrix proteins allows for storage and extended availability of gluten in the mucosa. New therapeutic approaches aim at proteolytic destruction of immunodominant gliadin peptides that are resistant to intestinal enzymes by bacterial prolyl endopeptidases, the inhibition of tTG activity with highly specific enzyme inhibitors or at HLA-DQ2/DQ8 blocking peptide analogues.     

温度和pH对重组人血管内皮抑制素脱酰胺的影响

目的利用液质联用技术研究温度和pH对重组人血管内皮抑制素中天冬酰胺(Asn)脱酰胺过程的影响。方法在不同的温度和pH值条件下,对重组人血管内皮抑制素样品溶液进行孵化,酶解处理后利用液质联用技术识别发生脱酰胺的多肽并测定其含量随样品放置温度和pH条件的变化。结果质谱分析结果表明酶解产物中多肽SVWHGSDPNGR序列中的Asn发生了脱酰胺化,Asn残基生成了天冬氨酸和异天冬氨酸残基,其他位置的Asn均未检测出明显的脱酰胺化;实验获得了不同温度和pH条件下重组人血管内皮抑制素中Asn脱酰胺化的速率及动力学常数;Asn脱酰胺过程随着温度和溶液pH值的升高,Asn脱酰胺速率上升。结论重组人血管内皮抑制素中天冬酰胺的脱酰胺过程受储存温度和pH值影响,研究药用蛋白质在不同条件下的脱酰胺过程对于筛选合适的储存条件、减少活性损失具有重要意义。     

Identification of a Gliadin T-Cell Epitope in Coeliac Disease: General Importance of Gliadin Deamidation for Intestinal T-Cell Recognition

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified γ-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.     

Tissue transglutaminase enhances collagen type II‐induced arthritis and modifies the immunodominant T‐cell epitope CII260‐270

The calcium‐dependent enzyme tissue transglutaminase (tTG) is associated with diverse biological functions, such as induction of apoptosis, modeling of the extracellular matrix, receptor‐mediated endocytosis, cell growth and differentiation, cell adhesion and signal transduction. Also, it may deamidate glutamine residues to glutamic acid and catalyze cross‐linking of proteins. In this study, we have investigated the impact of tTG for posttranslational modifications and cross‐linking of the immunodominant T‐cell epitope CII260‐270 and their effects on the collagen‐induced arthritis, an animal model for rheumatoid arthritis. By using mass spectrometry analysis and hybridoma assays, we have demonstrated that tTG could perform both types of modifications (deamidation and cross‐link formation) on the immunodominant T‐cell epitope CII259‐273. Replacement of the glutamine at position 267 with glutamic acid leads to a decreased binding affinity to MHC II. T cells recognized both non‐modfied (Q²⁶⁷) and modified (E²⁶⁷) CII259‐273‐peptides. We also show that administration of tTG leads to increased incidence, severity and histopathological manifestations of collagen‐induced arthritis in mice. Moreover, we conclude that both processes, deamidation and cross‐linking, are involved in the tTG‐catalyzed reactions, and in vivo administration of tTG enhances arthritis severity and joint destruction in mice.     

Identification of the mycobacterial DNA-binding protein 1 region which suppresses transcription in vitro

We recently identified a novel DNA-binding protein from Mycobacterium bovis bacillus Calmette-Guérin (BCG), termed mycobacterial DNA-binding protein 1 (MDP1). MDP1 inhibited the in vitro syntheses of DNA, RNA and protein, and reduced growth rates of bacteria transformed with MDP1 genes. In this study, we examined the DNA binding regions of MDP1 by using a set of synthetic peptides. One dominant region was determined on peptide 4 composed of amino acids, at positions 31-50. The peptide 4 inhibited syntheses of both DNA and RNA in vitro. The critical amino acids residues for these functions were analysed utilizing synthetic peptides substituted with Ala. This domain was perfectly conserved in MDP1 homologues in mycobacteria, but not observed in other known DNA binding proteins. These results indicate mycobacteria possess a unique nuclear protein, which might be involved in growth regulation of these organisms.     

Autoepitope-mapping of the U1-70K Protein with Human–DrosophilaChimeric Proteins

The 70K protein is the major autoantigen for anti-RNP autoantibodies directed against the U1 small nuclear ribonucleoprotein complex particle. The U1-70K protein has been epitope-mapped by various groups, and a major antigenic region of about 70 amino acids has been found which overlaps with the RNA binding motif. Attempts to map the major antigenic region further with smaller cloned fragments or with peptides have been hampered by total loss of, or strongly reduced, antigenicity. Thus the major antigenic region is composed of conformational epitopes and a detailed analysis of particular epitopes has not been possible.In the present work, we examine the antigenicity of chimeric proteins assembled from the highly conserved Drosophila melanogaster 70K proteins grafted with human 70K segments. With this approach, the effects on antigenicity of exchanging particular segments can be assayed with the overall structure of the major antigenic domain kept relatively constant. Our results, supported by depletion experiments, show that residues 99–128 from the human protein are essential for recognition by both human and canine anti-RNP autoantibodies. These residues have to be presented in a manner that allows correct conformational interaction between the different protein domains.     

Antibodies to Wheat High-Molecular-Weight Glutenin Subunits in Patients with Celiac Disease

Background: Wheat gluten comprises gliadins and glutenins. The high-molecular-weight (HMW) glutenin subunits (GS)-1Dy10 are toxic for patients with celiac disease (CD). This study aimed to assess whether CD patients mount a serological response to HMW-GS-1Dy10. Methods: Recombinant HMW-GS-1Dy10 was deamidated using human recombinant tissue transglutaminase. MALDI-TOF was performed to compare the level of deamidation of glutamine residues between material before and after treatment. Enzyme-linked immunosorbent assays were developed. Sera from patients with untreated CD and gastrointestinal disease controls were tested and receiver operator characteristics were used to calculate cutoffs. Results: MALDI-TOF revealed a number of fragments matching known HMW-GS-1Dy10 sequences within both the deamidated and non-deamidated material. Evidence of deamidation of glutamine residues was found only within the human transglutaminase-treated material. Patients with untreated CD had significantly increased levels of serum antibodies to HMW-GS-1Dy10 compared to controls. Undeamidated HMW-GS-1Dy10 IgA antibodies had sensitivities and specificities of 72.5 and 78.26%, respectively. Deamidated HMW-GS-1Dy10 IgA antibodies had sensitivities and specificities of 76.8 and 65.2%. Undeamidated HMW-GS-1Dy10 IgG antibodies had sensitivities and specificities of 75.3 and 68.1%. Deamidated HMW-GS-1Dy10 IgG antibodies had sensitivities and specificities of 36.2 and 92.8%. Conclusion: Patients with untreated CD have raised antibody levels to HMW-GS-1Dy10, indicating the participation of these proteins in the adaptive immune response to gluten. Discrimination between CD patients and controls is not enhanced by deamidation of HMW-GS-1Dy10. Thus antibodies to these proteins are not useful markers for CD detection.     

Characterization of High Molecular Weight Amyloid A Proteins

Human amyloid A protein (AA) is usually composed of the NH2-terminal 76 amino acid residue of serum amyloid A protein (SAA), although lower and higher molecular weight fragments have been reported. We studied the primary structure of six AA proteins with molecular weights of 11 kDA-15kDA, as determined by SDS-PAGE. Automated Edman degradation of the intact purified proteins and sequence analysis of enzymatic peptides revealed that the AA proteins were composed of only 74 to 87 residues. Moreover, fragments of apolipoprotein E or histones 2a, 3 and 4 were associated with these AA molecules. Thus, AA heterogeneity may reflect diverse processing of the SAA precursor and a very close association with other proteins.     

N-terminal positively charged amino acids, but not their exact position, are important for apicoplast transit peptide fidelity in Toxoplasma gondii

The non-photosynthetic plastid – or apicoplast – of Toxoplasma gondii and other apicomplexan parasites is an essential organelle and promising drug target. Most apicoplast proteins are encoded in the nucleus and targeted into the organelle through the apicoplast's four membranes courtesy of a bipartite N-terminal leader sequence comprising of an endomembrane signal peptide followed by a plastid transit peptide. Apicoplast transit peptides, like plant plastid transit peptides, have no primary consensus, are variable in length and may be distinguishable only by a relative depletion of negative charged residues and consequent enrichment in basic residues. In this study we examine the role of charged residues within an apicoplast transit peptide in T. gondii by point mutagenesis. We demonstrate that positive charged residues, combined with the absence of negatively charged amino acids, are essential for apicoplast transit peptide fidelity, as also observed in P. falciparum. Furthermore, we show that positive charge is more important at the transit peptide's N-terminus than its C-terminus, and that the nature of the positive residue and the exact position of the N-terminal positive charge are not important. These results suggest that a simple, rule-based prediction for T. gondii transit peptides, similar to that successfully implemented for P. falciparum should help to identify apicoplast proteins and facilitate the identification of drug targets in this important human pathogen.     

Presentation of insulin and insulin A chain peptides to mouse T cells: involvement of cysteine residues.

The requirements for insulin presentation and recognition by A alpha b A beta b- and A alpha b A beta k-restricted mouse T cells were studied using a variety of derivatives of the insulin A chain. It was found that A chain peptides with irreversibly blocked Cys residues are non-stimulatory for the T cells. This suggests that at least one of the Cys residues is essential for recognition. On the other hand, all A chain peptides containing Cys residues modified in a way reversible by reaction with thiols are stimulatory yet differ in antigenic potency. All these A chain derivatives including a 14 amino acid fragment require uptake by antigen presenting cells (APC) for efficient presentation. Differences in stimulatory potency between the A chain peptides derived from the same insulin appear to be mainly due to the efficiency of uptake and/or processing by APC. Based on these findings we propose that processing in the case of insulin and its A chain derivatives involves the reductive deblocking of Cys residues or the rearrangement of disulfide bonds apart from a possible proteolytic cleavage. The same may apply to other proteins if Cys residues in the presented peptides are important for the interaction with Ia or the T cell receptor.     

The amino acid sequence determination of a granulin and polyhedrin from two baculoviruses infecting Agrotis segetum.

The amino acid sequence of Agrotis segetum Granulosis virus (AsGV) granulin and A. Segetum nuclear polyhedrosis virus (AsNPV) polyhedrin was determined by sequencing tryptic and chymotryptic peptides from reduced and carboxymethylated proteins and tryptic fragments of oxidized and maleylated granulin. The comparison of the established peptide structures with the primary structures of other occlusion body proteins from related baculoviruses was also used for the polypeptide chains' reconstruction. The polypeptide chains of AsGV granulin and AsNPV polyhedrin comprise 247 and 246 amino acid residues, respectively. The proteins possess a high percentage of homology in their primary structures (63%).     

Some tryptic peptides of bromovirus proteins

The action of trypsin on brome mosaic virus (BMV) at pH 8 resulted in the dissociation of the virus particles into an 8 S component which contained two proteins, I and F. Protein I was capable of reassembly at pH 8 or 5 into 16-nm spherical particles; protein F formed these particles only at pH 5. SDS-polyacrylamide-gel electrophoresis and amino acid analysis indicated that protein F was about 23 and protein I about 18 amino acid residues smaller than native BMV protein. Six peptides, a cleavage product of the acetylated N-terminal peptide, free arginine, and eight other peptides containing more than one basic amino acid were isolated and their amino acid compositions and N-terminal residues were determined. The BMV peptides were similar or identical to peptides isolated after the limited proteolysis of cowpea chlorotic mottle virus protein at the N-terminus. The sequences of these portions of both viral proteins are probably very similar and probably function as sites of RNA-protein interaction. Peptides isolated from a total tryptic digestion of broad bean mottle virus protein were unlike peptides cleaved in limited proteolysis of BMV and cowpea chlorotic mottle virus.     

Association of Lyn tyrosine kinase to the GM-CSF and IL-3 receptor common betac subunit and role of Src tyrosine kinases in DNA synthesis and anti-apoptosis

BACKGROUND: After GM-CSF or IL-3 stimulation, the activation of JAK2 tyrosine kinase and members of the Src family of tyrosine kinases takes place, followed by phosphorylation of betac tyrosine residues and the recruitment of SH2 containing molecules to the receptor complex. The exact role of Src kinases such as Lyn in this and other downstream signal transduction events remains unclear. RESULTS: We investigated the association of Lyn kinase with betac using synthetic peptides derived from the eight betac tyrosine residues and the Box 1 motif. We found that Lyn kinase GST fusion proteins bind to peptides corresponding to the membrane proximal region of betac and to peptides containing specific betac derived phosphorylated tyrosine residues. We also determined that betac tyrosine residues Y1,2 as well as Y7 and Y8 can act as substrates of Lyn. We further analysed the role of the Src kinases in DNA synthesis and anti-apoptosis downstream of GM-CSF by using the Src kinase inhibitor PP1 in murine BA/F3 cells stably expressing a series of mutant betac receptors. CONCLUSIONS: Lyn binds to betac derived peptides through multiple interactions, and may play an important role in betac phosphorylation. Src family kinases also play an essential role in GM-CSF mediated DNA synthesis, as well as an important role in anti-apoptosis in response to GM-CSF.     

Simian virus 40 Vp2/3 small structural proteins harbor their own nuclear transport signal.

We have used a microinjection approach to identify a domain of the simian virus 40 (SV40) structural proteins Vp2 and Vp3(Vp2/3) responsible for their nuclear transport. By using both synthetic peptides, containing small regions of Vp2/3 conjugated to bovine serum albumin (BSA), and beta-galactosidase-Vp3 fusion proteins, we have narrowed this nuclear transport signal (NTS) to 9 amino acids (198 to 206 of Vp3 or 316 to 324 of Vp2), Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu. The porter proteins carrying the NTS or mutant NTS were microinjected into the cytoplasm of TC7 cells and their subcellular localization following the subsequent incubation period was determined immunologically using anti-BSA IgG or anti-beta-galactosidase. The 9-residue NTS peptide localized BSA into the nucleus of injected cells, changing lysine-202 to threonine or valine abolished this accumulation while changing arginine-204 to lysine did not grossly affect transport. A peptide containing the carboxyl-terminal 13 residues of Vp3 failed to localize BSA to the nucleus. Several single or double point mutations at Vp3 residues 202 and 204 have been introduced by site-directed mutagenesis. Vp3 residues 194-234, containing either a wild-type or mutated sequence at 202 and/or 204, were expressed in Escherichia coli as Vp3-beta-galactosidase fusion proteins. Addition of the carboxyl-terminal 40 residues, but not an internal 150 residues, to otherwise cytoplasmic beta-galactosidase promoted entry of the fusion protein into the nucleus. Changing lysine-202 into threonine, valine, or methionine abolished this nuclear accumulation as did changing arginine-204 into lysine. A double mutant at both positions was also blocked. We have also observed that the lectin wheat germ agglutinin inhibits the nuclear accumulation of BSA carrying the Vp2/3 NTS while the lectin concanavalin A had no effect. These data indicate that even small nuclear proteins can contain NTS's which most likely utilize a mechanism for nuclear import similar to that described for other larger proteins.     

Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303

A previous pool sequence analysis showed that HLA-A*3101 and HLA-A*3303 binding peptides have the same anchor residues at P2 and the C-terminus, the only difference being that HLA-A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA-S transfectants expressing HLA-A*3101 and human beta2-microglobulin, we tested the binding of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues to HLA-A*3101. One hundred of these peptides (43.1%) bound to HLA-A*3101, confirming that these residues are also anchors for HLA-A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA-A*3303 binding peptides, we detected no significant difference in HLA-A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA-A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA-A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA-A*3101 and HLA-A*3303 is determined by the polarity of the P1 anchor residue.